PROJECT SUMMARY There is a fundamental gap in understanding how Cryptosporidium sporozoites interact with host cells to initiate invasion and replication. It will be necessary to address this gap in order to explain distinguishing aspects of Cryptosporidium biology and host specificity. The long-term goal is to reduce the disease burden of cryptosporidiosis by understanding and disrupting the mechanisms by which the pathogen invades and replicates in human host cells. The objective in this proposal is to determine the role of host cell glycans during the transition of Cryptosporidium from an invasive sporozoite to a replicative trophozoite. The central hypothesis is that a specific presentation of galactose/N-acetylgalactosamine (Gal/GalNAc) mediates both attachment/invasion and trophozoite development, and that trophozoite development must coincide with attachment/invasion for a successful infection. This hypothesis is predicated on preliminary experiments undertaken in the applicants' laboratories. The rationale for the proposed research is that by identifying the precise structure and spatial presentation of host glycans recognized by human pathogenic Cryptosporidium species, molecules can be designed that interfere with the process of invasion and replication. The hypothesis will be tested by pursuing three specific aims: 1] to determine the extent to which glycan structure and spatial presentation affects trophozoite development; 2] to determine the extent to which glycan structure and spatial presentation at the host cell surface affects attachment/invasion; and 3] to determine the fate and biological significance of extracellular trophozoites during an infection. Under the first aim, synthetic glycopolymers with precisely controlled presentations of glycans will be used to identify the glycan structures that that trigger trophozoite development in the two major human pathogenic species of Cryptosporidium. Trophozoite development will be determined by examining parasites for rounding and nuclear division using microscopy. Under the second aim, lipid terminated synthetic glycopolymers will be incorporated into the membranes of HCT-8 cells ? a human adenocarcinoma cell line ? and susceptibility to infection by Cryptosporidium will be assessed using an immunofluorescence assay to separately detect attached and invaded parasites. Under the third aim, the host cell response to a Cryptosporidium infection will be determined by examining changes in the transcriptome and secretion of the trophozoite trigger. The fate of extracellular trophozoites will be assessed using microscopy to detect attachment/invasion and quantitative RT-PCR to detect markers of life cycle progression. The proposed research is innovative in its use of synthetic glycopolymers to identify the precise glycan structures affecting attachment, invasion, and replication of an intestinal parasite. The proposed research is significant because it has the potential to transform understanding of Cryptosporidium biology and accelerate the discovery of drug and vaccine targets.